Dynamic signature of activity-stability tradeoff in lactamase evolution

Our ability to understand protein evolution hinges on understanding how evolutionary landscapes are shaped at the fundamental protein level. Using TEM-1 β-lactamase we show that molecular traits related to the statistical ensemble nature of protein structure contribute to broader substrate specificity, active site-scaffold communication, and the selection of stabilizing substitutions. During the evolution of cefotaxime resistance, the initial mutation reorganizes the active site, introducing a new function conformation. Secondary substitutions improve catalytic efficiency by redistributing the ensemble and restoring a significant population of the original conformation, rather than by stabilizing the new conformation. Stability defects associated with initial mutations are not evenly disseminated but are clustered at specific distal scaffold elements. The capacity of mutants to independently modulate the populations of individual active site walls and scaffold regions through narrow residue networks, produces conformational epistasis and a combinatorial set of cefotaximase states, which enables local compensation of scaffold defects.